Our goal is to generate a molecular-resolution picture of the mechanisms underlying the control of membrane traffic in cells. The clathrin dependent pathways are of particular interest, as they are general paradigms for understanding membrane vesicular traffic. We use various imaging techniques, including cryoEM 3-D single-particle image reconstruction, cryo-electron tomography, x-ray crystallography, and fluorescence microscopy, to visualize components of the trafficking machinery, and we use the structural data to inform biochemical and mechanistic studies. Our focus during the coming grant period will be on the regulation of clathrin coat assembly and disassembly and on novel interactions of the heterotetrameric adaptor complexes (APs). There are three sets of aims. (1) We will study the molecular mechanism of uncoating by Hsc70 and its co-chaperon, auxilin, using cryoEM reconstructions, structure-based mutagenesis in vitro and in vivo, and single-object fluorescence microscopy. We will also use a structure- based approach to determine the role in uncoating of specific lipid head-group recognition by the PTEN-like domain of auxilin. (2) We will study a new aspect of cargo recognition by the clathrin adaptor complexes, involving recognition of Dishevelled, a component of the Wnt signaling pathway. (3) We will dissect the steps of coat assembly on membranes, using a combination of biochemical methods, high-resolution structure analysis, and cryo-electron tomography. By the end of this grant period, we aim to be able to study the dynamics of clathrin coat assembly and disassembly in a reconstituted system, in parallel with our work on coat dynamics in living cells.